Detection methods for cryptosporidium

ABSTRACT

PCT No. PCT/AU96/00387 Sec. 371 Date Mar. 20, 1998 Sec. 102(e) Date Mar. 20, 1998 PCT Filed Jun. 25, 1996 PCT Pub. No. WO97/02281 PCT Pub. Date Jan. 23, 1997The invention provides a purified and isolated Cryptosporidium DNA sequence comprising the nucleotide sequence: GATGGTACTGGATAGATAGTGGAAGTCCCGTATCAGTTCGAGATTCTGAAATTA ATTGGACATCAAGTTATAAAGCAAGCTGGTTATTAAGATTCAAATTTCCCTTTGA AAAGTGTGGCTTTTTTGATATTGGAGGGTTAGGAAGAAGGTT plus methods and kits for detecting and/or identifying the presence of Cryptosporidium.

The present invention relates to a method for detecting microorganismsof the genus Cryptosporidium and more particularly Cryptosporidiumparvum.

The protozoan parasite Cryptosporidium parvum is recognised as animportant cause of diarrhoeal illness primarily in infants and youngchildren, (although immunologically healthy adults are susceptible) andis associated with persistent diarrhoea and severe illness inmalnourished children. It is also a serious opportunistic pathogen inimmunocompromised individuals, causing severe and unremitting diarrhoeathat is often intractable to therapy. Chronic cryptosporidiosis isreported in as many as 10% of persons with AIDS in the United States andthere are currently no effective therapeutic strategies for treatingCryptosporidium infection.

Waterborne transmission of this enteric parasite is a major concern. Theinfective stage (oocyst) of Cryptosporidium is transmitted by thefaecal-oral route, with infected individuals excreting Cryptosporidiumoocysts. Animals as well as humans may serve as sources of environmentalcontamination and human infection. The oocyst is environmentally stableand is able to survive and penetrate routine wastewater treatment and isresistant to inactivation by drinking water disinfectants. There areseveral species of Cryptosporidium but Cryptosporidium parvum isbelieved to cause the majority of mammalian infections. Cryptosporidiumparvum oocysts are resistant to chlorination procedures normally usedfor water treatment, and contamination of water supplies can causemassive outbreaks of the disease such as the 1994 outbreak ofcryptosporidiosis in Milwaukee resulting in diarrhoeal illness in anestimated 403,000 people.

In the absence of effective drugs to treat this ubiquitous infection,the control and clinical management of cryptosporidiosis depends uponrapid, accurate and sensitive diagnosis of the presence of the parasite,both in clinical specimens and environmental samples.

Clinical diagnosis of Cryptosporidium is time consuming, insensitive andgenerally requires the skills of highly trained operators. It hasrecently been reported that the detection limits of conventionaldiagnostic techniques for Cryptosporidium were as low as 50,000 oocystsper gram of faeces and that mean oocyst losses ranged from 51.2% to99.6%. Further, the most commonly used coprodiagnostic techniques mayfail to detect cryptosporidiosis in many immunocompromised andimmunocompetent individuals. Immunological-based detection methods usingimmunofluorescence assays, enzyme-linked-immunosorbent andimmunofluorescent-based diagnostic tests have been developed, several ofwhich are now commercially available. Enzyme-linked immunoassays,although quick and easy to perform, generally show low sensitivityranging from 3×10⁵ to 1×10³ oocysts per gram of faeces and monoclonalantibodies have the ability to bind to other microorganisms, i.e., tostain nonspecifically. In addition, Cryptosporidium isolates have beenshown to exhibit a great deal of antigenic variability and thereforediagnostic antibodies may not recognise all isolates.

Environmental detection of Cryptosporidium generally involves filteringlarge volumes of water and examining it microscopically forCryptosporidium oocysts by various staining or immunolabellingtechniques. However, the efficiency of oocyst recovery may be as low as1.3 to 5.5%. Recently, an alternative means of harvesting oocysts bycalcium carbonate flocculation has been described with improved recoveryranging from 68% to >80%. Specialised flow cytometry and cell sortingtechniques have also been developed to detect oocysts in water sampleswith greater sensitivity than conventional fluorescence microscopy.Although these methods are significantly more sensitive and considerablyfaster than conventional methods, they are costly and still require theskills of highly trained technical operators.

The development of the polymerase chain reaction (PCR) has permittedspecific and sensitive detection of pathogens for clinical diagnosis andenvironmental monitoring. Diagnostic PCR primers have been described forthe detection of Cryptosporidium. However, these primers suffer from alack of sensitivity and are only able to detect at best approximately200 Cryptosporidium oocysts reliably under optimum conditions. Further,most of the primers selected to date have only been tested on a smallnumber of Cryptosporidium isolates and none of them have been testeddirectly on faeces. Thus, there exists a need for a sensitive detectionmethod which is capable of identifying the presence of Cryptosporidiumin faeces and environmental samples.

Given the severity and untreatable nature of Cryptosporidium infectionin persons with AIDS, early detection of cryptosporidial infection inHIV-infected or AIDS patients who may be shedding low numbers of oocystsbecomes increasingly important. A rapid, sensitive assay requiringlittle or no expertise on the part of the operator would be of greatbenefit in the early detection of asymptomatic or mild cryptosporidialinfection in AIDS patients. It would improve clinical management of thedisease with the option of initiating chemotherapy before the onset ofsymptoms, which may result in fewer cases progressing to severe, andoften chronic, infections of this parasite.

The present invention provides nucleotide sequences which may beutilised in diagnostic assays to analyse samples for environmentalcontamination by Cryptosporidium oocysts and for the diagnosis ofCryptosporidium infections in patients.

Thus, the invention consists of a purified and isolated CryptosporidiumDNA sequence comprising the nucleotide sequence:

    GATGGTACTGGATAGATAGTGGAAGTCCCGTATCAGTTCGAGATTCTGAAA                            TTAATTGGACATCAAGTTATAAAGCAAGCTGGTTATTAAGATTCAAATTTC                           CCTTTGAAAAGTGTGGCTTTTTTGATATTGGAGGGTTAGGAAGAAGGCCGT                           GTTGGCTTATAGATTCTGAGCTTTCTTGTGCAGTTTGTGGTACAGTAGCTT                           ATGATTCTGGTGGGCTGAATCCCAATAAATATTCAGAGCTAATTAAGCAGA                           CTGATGAAATTATTAGTAAAGAGCCAAAGCTTGATCTTCCAGGTTACAATA                           ATTTGAACTGTACAGATGCTTGGGAGAATAATTTATCAGTTGGTCTTTGTC                           AAAATGTCTCAAATATCCTGGACTCAGCTTGGAGCTCGTATCAGAGTTCGT                           TAAACTTTCCTAGTATCAACTTTAACTGGAAAGAGGATTCAACTAACGAAG                           GAGGGGACCAAGTTTACCATAATTCTTATTTGGATCTTCCAAGGTATAAGC                           AGAAGAAAACATTTTATTGGGATCAGGATCCAGGTACTATTCCAGCTTTGT                           CTGATGAAATGAAGCTCATTGGTTTAAGCGCTCAACCAACATACCATCCTT                           TGGATAGAAGCTCATCTGGAAGTTTTGAGTCTGATAGTACAGAATCCGGGC                           GTGCGAATGAAGAAAGAAACGATAC                                                

Preferably, the present invention consists of a method for detectingand/or identifying the presence of Cryptosporidium genomic material in asample, said method comprising the steps of: selecting at least a primeror probe derived from the above mentioned nucleotide sequence; and thenusing that primer or probe to detect and/or identify the presence ofCryptosporidium genomic material.

From the above nucleotide sequence, oligonucleotides can be preparedwhich hybridise with the Cryptosporidium genome. The oligonucleotidesmay be used either as a primer(s) or as a probe(s) to detect theCryptosporidium genome. Preferably, the primer(s) or probe(s) arespecific for microorganisms of the species Cryptosporidium parvum.

The primer(s) or probe(s) for Cryptosporidium are preferably of a lengthwhich allows for the specific detection of such microorganisms.Primer(s) or probe(s) which are 5 to 8 nucleotides in length should besuitable for detecting the Cryptosporidium genome. Preferably, sequencesof 10 to 50 nucleotides may be used as primer(s) or probe(s). Moreparticularly, sequences of about 15 to 25 nucleotides may be used in theidentification protocols, and about 20 to 24 nucleotides appear optimal.

Primer(s) or probe(s) can be selected and prepared using routinemethods, including automated oligonucleotide synthetic methods. Acomplement to any unique portion of the above nucleotide sequence may beused as a primer(s) or probe(s) provided that it specifically binds othe Cryptosporidium genome. When used as primer(s) or probe(s) completecomplementarity is desirable, though it may be unnecessary as the lengthof the fragment is increased. Among useful primer(s) or probe(s) forsetecting and/or identifying Cryptosporidium isolates are, for example,the following sequences:

    (i)     5' GGTACTGGATAGATAGTGGA 3'                                                      - (ii) 5' TCGCACGCCCGGATTCTGTA 3'                                      - (iii) 5' AGTCCCGTATCAGTTCGAGA 3'                                            - (iv) 5' ACTGGATAGATAGTGGAAGT 3'                                             - (v) 5' TTTCTTTCTTCATTCGCACG 3'                                              - (vi) 5' GTGGAAGTCCCGTATCAGTC 3'                                             - (vii) 5' ACGCCCGGATTCTGTACTAT 3'                                            - (viii) 5' GATAGATAGTGGAAGTCCCG 3'                                           - (ix) 5' ACGCCCGGATTCTGTACTAT 3'                                             - (x) 5' CTGAAATTAATTGGACATCA 3'                                              - (xi) 5' GTACTATCAGACTCAAAACT 3'                                             - (xii) 5' GTGGTACTGGATAGATAGTG 3'                                            - (xiii) 5' GTATCGTTTCTTTCTTCATT 3'                                           - (xiv) 5' TGGTACTGGATAGATAGTGG 3'                                            - (xv) 5' TATCGTTTCTTTCTTCATTC 3'                                             - (xvi) 5' TAGATAGTGGAAGTCCCGTA 3'                                            - (xvii) 5' TCTTCATTCGCACGCCCGGA 3'                                           - (xviii) 5' ATAGTGGAAGTCCCGTATCA 3'                                          - (xix) 5' TTTCTTCATTCGCACGCCCG 3'                                            - (xx) 5' CTGGATAGATAGTGGAAGTC 3'                                             - (xxi) 5' CGTTTCTTTCTTCATTCGCA 3'                                            - (xxii) 5' TAATTGGACATCAAGTATAA 3'                                           - (xxiii) 5' GTACTATCAGACTCAAAACT 3'                                          - (xxiv) 5' TCTGAAATTAATTGGACATC 3'                                           - (xxv) 5' CTTCCAGATGAGCTTCTATC 3'                                            - (xxvi) 5' GGTGGTACTGGATAGATAGT 3'                                           - (xxvii) 5' GGTATCGTTTCTTTCTTCAT 3'                                          - (xxviii) 5' GAGATTCTGAAATTAATTGG 3'                                         - (xxix) 5' GTTGGCTTATAGATTCTGAGC 3'                                          - (xxx) 5' GGTTATTAAGATTCAAATTTCC 3'                                          - (xxxi) 5' TCCCGTATCAGTTCGAGATTCTG 3'                                        - (xxxii) 5' CGAACTCTGATACGAGCTCCAAGC 3'                                      - (xxxiii) 5' ATTCGAGATTCTGAAATTAATTGG 3'                                     - (xxxiv) 5' GAATAGTACCTGGATCCTGATCCC 3'                                      - (xxxv) 5' GATATTGGAGGGTTAGGAAGAAGG 3'                                       - (xxxvi) 5' CTGTACAGTTCAAATTATTGTAACC 3'                                     - (xxxvii) 5' GACTGATGAAATTATTAGTAAAGAGC 3'                                   - (xxxviii) 5' CCTCCTTCGTTAGTTGAATCCTC 3'                                     - (xxxix) 5' TCGCACGCCCGGATTCTGTA 3'                                          - (xl) 5' CAGTTCAAATTATTGTAGCC 3'                                             - (xli) 5' GTTCGAGATTCTGAAATTAATTGG 3'                                        - (xlii) 5' GTCCCGTATCAGTTCGAGATTCTG 3'                                       - (xliii) 5' GGAGGGTTAGGAAGAAGGCCGTG 3'                                       - (xliv) 5' GCTTGGGAGAATAATTTATCAG 3'                                         - (xlv) 5' GGGATCAGGATCCAGGTACTATTC 3'                                        - (xlvi) 5' GTATCGTTTCTTTCTTCATTCGC 3'                                        - (xlvii) 5' GGACCAAGTTTACCATAATTC 3'                                         - (xlviii) 5' GGAGAATAATTTATCAGTTGGTC 3'                                      - (xlix) 5' CAAGGTATAAGCAGAAGAAAAC 3'                                         - (l) 5' CGCACGCCCGGATTCTGTACTATC 3'                                          - (li) 5' ATGTCTCAAATATCCTGGACTCAG 3'                                         - (lii) 5' GTACTGGATAGATAGTGGAAGTC 3'                                         - (liii) 5' CACGGCCTTCTTCCTAACCCTCC 3'                                        - (liv) 5' GGAAGTCCCGTATCAGTTCGAG 3'                                   

Before the above probe(s) or primer(s) are used to detect and/oridentify Cryptosporidium isolates in diagnostic methods such as thosediscussed herein, the sample to be analysed, such as a faecal sample, ispreferably treated to extract the nucleic acid material containedtherein. The resulting nucleic acid material from the sample may then besubjected to gel electrophoresis or other size separation techniques;the nucleic acid material may be blotted without size separation;alternatively, the sample may be tested without being subjected to suchtechniques. Whether size separation is employed in the identificationprotocol will depend on the type of assay being used. For example, sizeseparation may be useful in hybridization assays.

Depending on the detection method which is employed to detect and/oridentify the presence of Cryptosporidium isolates in a sample, theprobe(s) or primer(s) may be labelled. Suitable labels and methods forlabelling probes and primers are known in the art. For example, probesor primers may be labelled using radioactive deoxynucleotide labelsincorporated by nick translation or end labelling, biotin labels,fluorescent labels or chemiluminescent labels may also be used.Alternatively, Cryptosporidium specific polynucleotides may be detectedon agarose or poly acrylamide gels using, for example, ethidiumbromide/UV visualisation or by silver staining techniques.

In one detection method, Cryptosporidium specific polynucleotidesextracted from the sample may be treated with a labelled probe underhybridisation conditions of suitable stringencies. Usually highstringency conditions are desirable to prevent false positives. Thestringency of hybridisation is determined by a number of factors duringhybridisation and during the washing procedure, including temperature,ionic strength, length of time and concentration of reactants. A personof ordinary skill in the art would understand how these factors may beused together to modify the stringency of hybridisation.

Generally, it is expected that Cryptosporidium DNA will be present insamples from infected individuals and particularly in environmentalsamples at low concentrations. This level may dictate the need foramplification of the nucleic acids before they can be detected. Suchamplification techniques are known in the art.

A method that is particularly preferred for detecting CryptosporidiumDNA is based on a PCR type test wherein a set of primers which arehighly specific for Cryptosporidium DNA are used to amplifyCryptosporidium DNA present in a sample. The presence of the resultantproduct can then be detected using, for example, ethidium bromide/UVvisualisation or by silver staining techniques. Alternatively,colourimetric detection of the PCR products using biotinylated primerscould be employed to save time and to eliminate the need for agarose gelelectrophoresis. Such an assay could also be modified to suit a 96 wellmicrotitre format for bulk processing of samples.

Thus, in one embodiment the invention provides a method of detectingand/or identifying microorganisms of the genus Cryptosporidiumcomprising the steps of:

(i) selecting at least a set of primers from the above nucleotidesequence which are specific for Cryptosporidium DNA;

(ii) mixing the primers with a sample suspected of containingCryptosporidium DNA;

(iii) amplifying any DNA to which the primers in step (ii) anneal by thepolymerase chain reaction; and

(iv) detecting the presence of the product of step (iii).

Although the above method has general application to one or more speciesof Cryptosporidium, preferably the primers which are selected in step(i) are highly specific for Cryptosporidium parvum.

Primer pairs which may be suitable for detecting Cryptosporidium parvumare preferably selected from the following sequences. In each primer setdescribed the first mentioned primer represents the forward primer andthe second mentioned primer represents the reverse primer.

    (i)     5' ACTGGATAGATAGTGGAAGT 3'                                               -  5' TTTCTTTCTTCATTCGCACG 3'                                                 - (ii) 5' GTGGAAGTCCCGTATCAGTC 3'                                             -  5' ACGCCCGGATTCTGTACTAT 3'                                                 - (iii) 5' GATAGATAGTGGAAGTCCCG 3'                                            -  5' ACGCCCGGATTCTGTACTAT 3'                                                 - (iv) 5' CTGAAATTAATTGGACATCA 3'                                             -  5' GTACTATCAGACTCAAAACT 3'                                                 - (v) 5' GTGGTACTGGATAGATAGTG 3'                                              -  5' GTATCGTTTCTTTCTTCATT 3'                                                 - (vi) 5' TGGTACTGGATAGATAGTGG 3'                                             -  5' TATCGTTTCTTTCTTCATTC 3'                                                 - (vii) 5' TAGATAGTGGAAGTCCCGTA 3'                                            -  5' TCTTCATTCGCACGCCCGGA 3'                                                 - (viii) 5' ATAGTGGAAGTCCCGTATCA 3'                                           -  5' TTTCTTCATTCGCACGCCCG 3'                                                 - (ix) 5' CTGGATAGATAGTGGAAGTC 3'                                             -  5' CGTTTCTTTCTTCATTCGCA 3'                                                 - (x) 5' TAATTGGACATCAAGTATAA 3'                                              -  5' GTACTATCAGACTCAAAACT 3'                                                 - (xi) 5' TCTGAAATTAATTGGACATC 3'                                             -  5' CTTCCAGATGAGCTTCTATC 3'                                                 - (xii) 5' GGTGGTACTGGATAGATAGT 3'                                            -  5' GGTATCGTTTCTTTCTTCAT 3'                                                 - (xiii) 5' GGTACTGGATAGATAGTGGA 3'                                           -  5' TCGCACGCCCGGATTCTGTA 3'                                                 - (xiv) 5' GAGATTCTGAAATTAATTGG 3'                                            -  5' CCTCCTTCGTTAGTTGAATCC 3'                                                - (xv) 5' GTTGGCTTATAGATTCTGAGC 3'                                            -  5' CAGTTCAAATTATTGTAGCC 3'                                                 - (xvi) 5' GAGATTCTGAAATTAATTGG 3'                                            -  5' CAGTTCAAATTATTGTAACC 3'                                                 - (xvii) 5' GTTGGCTTATAGATTCTGAGC 3'                                          -  5' CCTCCTTCGTTAGTTGAATCC 3'                                                - (xviii) 5' TAATTGGACATCAAGTTATAAAGC 3'                                      -  5' GGAAGATCCAAATAAGAATTATGG 3'                                             - (xix) 5' GGTTATTAAGATTCAAATTTCC 3'                                          -  5' CGAACTCTGATACGAGCTCCAAGC 3'                                             - (xx) 5' TCCCGTATCAGTTCGAGATTCTG 3'                                          -  5' CGAACTCTGATACGAGCTCCAAGC 3'                                             - (xxi) 5' GTTCGAGATTCTGAAATTAATTGG 3'                                        -  5' CGAACTCTGATACGAGCTCCAAGC 3'                                             - (xxii) 5' TAATTGGACATCAAGTTATAAAGC 3'                                       -  5' CGAACTCTGATACGAGCTCCAAGC 3'                                             - (xxiii) 5' GGTTATTAAGATTCAAATTTCC 3'                                        -  5' CGAACTCTGATACGAGCTCCAAGC 3'                                             - (xxiv) 5' TCCCGTATCAGTTCGAGATTCTG 3'                                        -  5' GAATAGTACCTGGATCCTGATCCC 3'                                             - (xxv) 5' TAATTGGACATCAAGTTATAAAGC 3'                                        -  5' GAATAGTACCTGGATCCTGATCCC 3'                                             - (xxvi) 5' GGTTATTAAGATTCAAATTTCC 3'                                         -  5' GAATAGTACCTGGATCCTGATCCC 3'                                             - (xxvii) 5' TCCCGTATCAGTTCGAGATTCTG 3'                                       -  5' GGAAGATCCAAATAAGAATTATGG 3'                                             - (xxviii) 5' GTTCGAGATTCTGAAATTAATTGG 3'                                     -  5' GGAAGATCCAAATAAGAATTATGG 3'                                             - (xxix) 5' GGTTATTAAGATTCAAATTTCC 3'                                         -  5' GGAAGATCCAAATAAGAATTATGG 3'                                             - (xxx) 5' ATTCGAGATTCTGAAATTAATTGG 3'                                        -  5' GAATAGTACCTGGATCCTGATCCC 3'                                             - (xxxi) 5' GATATTGGAGGGTTAGGAAGAAGG 3'                                       -  5' CTGTACAGTTCAAATTATTGTAACC 3'                                            - (xxxii) 5' GACTGATGAAATTATTAGTAAAGAGC 3'                                    -  5' CCTCCTTCGTTAGTTGAATCCTC 3'                                              - (xxxiii) 5' GGTACTGGATAGATAGTGGAAG 3'                                       -  5' CCAGAATCATAAGCTACTGTACC 3'                                              - (xxxiv) 5' GTCCCGTATCAGTTCGAGATTCTG 3'                                      -  5' CCTCCTTCGTTAGTTGAATCCTC 3'                                              - (xxxv) 5' GGGATCAGGATCCAGGTACTATTC 3'                                       -  5' GTATCGTTTCTTTCTTCATTCGC 3'                                              - (xxxvi) 5' GCTTGGGAGAATAATTTATCAG 3'                                        -  5' CCTCCTTCGTTAGTTGAATCCTC 3'                                              - (xxxvii) 5' GGACCAAGTTTACCATAATTC 3'                                        -  5' GTATCGTTTCTTTCTTCATTCGC 3'                                              - (xxxviii) 5' GGAGAATAATTTATCAGTTGGTC 3'                                     -  5' GTATCGTTTCTTTCTTCATTCGC 3'                                              - (xxxix) 5' CAAGGTATAAGCAGAAGAAAAC 3'                                        -  5' CGCACGCCCGGATTCTGTACTATC 3'                                             - (xl) 5' ATGTCTCAAATATCCTGGACTCAG 3'                                         -  5' CGCACGCCCGGATTCTGTACTATC 3'                                             - (xli) 5' GTACTGGATAGATAGTGGAAGTC 3'                                         -  5' CACGGCCTTCTTCCTAACCCTCC 3'                                       

Particularly preferred primer pairs that may be used in a diagnosticmethod for detecting Cryptosporidium parvum are desirably selected fromthe following primer sets. In each primer set described the firstmentioned primer represents the forward primer and the second mentionedprimer represents the reverse primer.

    (i)    5' GGTACTGGATAGATAGTGGA 3'                                                -  5' TCGCACGCCCGGATTCTGTA 3'                                                 - (ii) 5' GAGATTCTGAAATTAATTGG 3'                                             -  5' CCTCCTTCGTTAGTTGAATCC 3'                                                - (iii) 5' GTTGGCTTATAGATTCTGAGC 3'                                           -  5' CAGTTCAAATTATTGTAGCC 3'                                                 - (iv) 5' GAGATTCTGAAATTAATTGG 3'                                             -  5' CAGTTCAAATTATTGTAACC 3'                                                 - (v) 5' GTTGGCTTATAGATTCTGAGC 3'                                             -  5' CCTCCTTCGTTAGTTGAATCC 3'                                                - (vi) 5' TAATTGGACATCAAGTTATAAAGC 3'                                         -  5' GGAAGATCCAAATAAGAATTATGG 3'                                             - (vii) 5' GGTTATTAAGATTCAAATTTCC 3'                                          -  5' CGAACTCTGATACGAGCTCCAAGC 3'                                             - (viii) 5' TCCCGTATCAGTTCGAGATTCTG 3'                                        -  5' CGAACTCTGATACGAGCTCCAAGC 3'                                             - (ix) 5' GTTCGAGATTCTGAAATTAATTGG 3'                                         -  5' CGAACTCTGATACGAGCTCCAAGC 3'                                             - (x) 5' TAATTGGACATCAAGTTATAAAGC 3'                                          -  5' CGAACTCTGATACGAGCTCCAAGC 3'                                             - (xi) 5' GGTTATTAAGATTCAAATTTCC 3'                                           -  5' CGAACTCTGATACGAGCTCCAAGC 3'                                             - (xii) 5' TCCCGTATCAGTTCGAGATTCTG 3'                                         -  5' GAATAGTACCTGGATCCTGATCCC 3'                                             - (xiii) 5' TAATTGGACATCAAGTTATAAAGC 3'                                       -  5' GAATAGTACCTGGATCCTGATCCC 3'                                             - (xiv) 5' GGTTATTAAGATTCAAATTTCC 3'                                          -  5' GAATAGTACCTGGATCCTGATCCC 3'                                             - (xv) 5' TCCCGTATCAGTTCGAGATTCTG 3'                                          -  5' GGAAGATCCAAATAAGAATTATGG 3'                                             - (xvi) 5' GTTCGAGATTCTGAAATTAATTGG 3'                                        -  5' GGAAGATCCAAATAAGAATTATGG 3'                                             - (xvii) 5' GGTTATTAAGATTCAAATTTCC 3'                                         -  5' GGAAGATCCAAATAAGAATTATGG 3'                                             - (xviii) 5' ATTCGAGATTCTGAAATTAATTGG 3'                                      -  5' GAATAGTACCTGGATCCTGATCCC 3'                                             - (xix) 5' GATATTGGAGGGTTAGGAAGAAGG 3'                                        -  5' CTGTACAGTTCAAATTATTGTAACC 3'                                            - (xx) 5' GACTGATGAAATTATTAGTAAAGAGC 3'                                       -  5' CCTCCTTCGTTAGTTGAATCCTC 3'                                              - (xxi) 5' GGTACTGGATAGATAGTGGAAG 3'                                          -  5' CCAGAATCATAAGCTACTGTACC 3'                                              - (xxii) 5' GTCCCGTATCAGTTCGAGATTCTG 3'                                       -  5' CCTCCTTCGTTAGTTGAATCCTC 3'                                              - (xxiii) 5' GGGATCAGGATCCAGGTACTATTC 3'                                      -  5' GTATCGTTTCTTTCTTCATTCGC 3'                                              - (xxiv) 5' GCTTGGGAGAATAATTTATCAG 3'                                         -  5' CCTCCTTCGTTAGTTGAATCCTC 3'                                              - (xxv) 5' GGACCAAGTTTACCATAATTC 3'                                           -  5' GTATCGTTTCTTTCTTCATTCGC 3'                                              - (xxvi) 5' GGAGAATAATTTATCAGTTGGTC 3'                                        -  5' GTATCGTTTCTTTCTTCATTCGC 3'                                              - (xxvii) 5' CAAGGTATAAGCAGAAGAAAAC 3'                                        -  5' CGCACGCCCGGATTCTGTACTATC 3'                                             - (xxviii) 5' ATGTCTCAAATATCCTGGACTCAG 3'                                     -  5' CGCACGCCCGGATTCTGTACTATC 3'                                             - (xxix) 5' GTACTGGATAGATAGTGGAAGTC 3'                                        -  5' CACGGCCTTCTTCCTAACCCTCC 3'                                       

If for example the forward and reverse PCR primers areGGTACTGGATAGATAGTGGA and TCGCACGCCCGGATTCTGTA respectively, a DNAfragment of approximately 668 nucleotides is produced upon amplificationof Cryptosporidium parvum DNA. Alternatively, if the forward and reversePCR primers are GAGATTCTGAAATTAATTGG and CCTCCTTCGTTAGTTGAATCCrespectively, a DNA fragment of approximately 426 nucleotides isproduced upon amplification of Cryptosporidium parvum DNA.

The methods described herein may be used to detect the presence orabsence of Cryptosporidium DNA. However, they provide little informationabout the viability or infective potential of microorganisms within asample. Thus, the detection method(s) described supra may be combinedwith one or more methods for testing Cryptosporidium viability. Suchmethods are widely known in the art. For example fluorogeneic vital-dyeassays (eg. using propidium iodide) or the ability of Cryptosporidium togrow in vitro or in vivo may be used to determine the likely infectivityof virulence of the samples tested. Care should, however, be used whenemploying such methods. Current protocols for concentrating oocysts suchas Percoll-sucrose gradients and sucrose density flotation may actually,selectively concentrate non-viable oocysts. Standard tests for viabilitysuch as fluorogeneic vital-dye assays may therefore be biased towardsdetection of non-viable oocysts. In addition, current protocols onlysample a proportion of the total water body and the viability of cystsand oocysts not detected remains undetermined.

While the present invention relates to nucleotide sequences ofCryptosporidium and methods for detecting and/or identifying thepresence of Cryptosporidium isolates, it will be appreciated that thesequences and method(s) may be made available in the form of a kit forthe detection of Cryptosporidium isolates. Preferably, the kit providesa means for detecting Cryptosporidium parvum isolates.

Thus, in one embodiment of the invention there is provided a kit ofdetecting and/or identifying the presence of Cryptosporidiummicroorgansims is a sample, the kit comprising: at least a probe or setof primers which are specific for a region of the genome ofCryptosporidium, wherein the probe or primers are selected from theabove mentioned nucleotide sequence.

Probes and primers can be packaged into diagnostic kits. Diagnostic kitsmay include the DNA probe or DNA primers which may be labelled;alternatively the probe or primers may be unlabelled and the ingredientsfor labelling the probe or amplifying the Cryptosporidium DNA using theprimers may be included in the kit. The kit may also contain othersuitably packaged reagents and materials needed for the particulardetection protocols. The kit may also contain, for example, standards,as well as instructions for using the detection kits. Particulardiagnostic kits may also contain the necessary reagents for conductingfluorogenic assays and/or for growing Cryptosporidium in cell culture.

The present invention will now be described by way of example only withreference to the following figures. It will be understood that alltemperature ranges and other such variables prescribed in the examplesare given as indicative only, and that parameters outside these limitsmay also provide useful results.

FIG. 1a represents an ethidium bromide stained 1% agarose gel showingspecificity of the 021 diagnostic primers for Cryptosporidium. Lane1=molecular weight marker; lane 2=C. parvum DNA; lane 3=G. duodenalisDNA; lane 4=human DNA; lane 5=faecal DNA; lane 6=Tritrichomonas foetusDNA; lane 7=C. serpentis DNA; lane 8=negative control (no DNA).Molecular weight marker was 100 bp ladder (Gibco BRL); kb=kilobases.

FIG. 1b shows the specificity testing of the CP1 primers. Lane1=molecular weight marker; lane 2=C. parvum DNA; lane 3=G. duodenalisDNA; lane 4=human DNA; lane 5=faecal DNA; lane 6=Tritrichomonas foetusDNA; lane 7=negative control (no DNA). Molecular weight marker as inFIG. 1a.

FIG. 2a represents an ethidium bromide stained 1% agarose gel showingproducts obtained from amplification performed on 13 of the 35Cryptosporidium parvum isolates examined using the 021 primers. Lane1=molecular weight marker; lane 2=L1; lane 3=H9; lane 4=C1; lane 5=H7;lane 6=H5; lane 7=H6; lane 8=H3; lane 9=H10; lane 10=H8; lane 11=H4;lane 12=H1; lane 13=C6; lane 14=H2; lane 15=negative control. Molecularweight marker as in FIG. 1a. Isolates H11-12; H15-H34; C1 and C7-9 werealso tested and produced the desired 668 bp band upon amplification(data not shown).

FIG. 2b illustrates parasite origin of the bands depicted in FIG. 2a.The gel depicted in FIG. 2a was blotted onto Hybond N⁺ (Amersham) andprobed with the internal oligonuleotide probes to confirm parasiteorigin of bands.

FIG. 3 represents an ethidium bromide stained 1% agarose gel showingproducts obtained from amplification performed on 28 of the 39Cryptosporidium isolates examined using the CP1 primers. Lane1=molecular weight marker; lane 2=H1; lane 3=H2; lane 4=H3; lane 5=H5;lane 6=H6; lane 7=H7; lane 8=H8; lane 9=H9; lane 10=H10; lane 11=H11;lane 12=H12; lane 13=15; lane 14=H16; lane 15=H17; lane 16=H18; lane17=H19; lane 18=H20; lane 19=H21; lane 20=H22; lane 21=H23; lane 22=H24;lane 23=H25; lane 24=H26; lane 25=C1; lane 26=C2; lane 27=C6; lane28=C7; lane 29=L1; lane 30=negative control. Isolates F1; F9; F10; F11;F20; F21; F22; F35; F36; F38 also amplified the correct 446 bp band.

FIG. 4a represents an ethidium bromide stained 1% agarose gel showingthe sensitivity of the 021 primers. Lane 1=molecular weight marker; lane2=1×10⁵ C. parvum oocysts; lane 3=1×10⁴ C. parvum oocysts; lane 4=1×10³C. parvum oocysts; lane 5=100 C. parvum oocysts; lane 6=10 C. parvumoocysts; lane 7=1 C. parvum oocys; and lane 8=negative control.Molecular weight marker as in FIG. 1a.

FIG. 4b represents an ethidium bromide stained 1% agarose gel showingthe sensitivity of the CP primers. Lane 1=molecular weight marker; lane2=1×10³ C. parvum oocysts; lane 3=100 C. parvum oocysts; lane 4=10 C.parvum oocysts; lane 5=1 C. parvum oocyst; lane 6=negative control (noDNA). Molecular weight marker as in FIG. 1a.

FIG. 5a represents an ethidium bromide stained 1% agarose gel showingdirect amplification of Cryptosporidium DNA from faeces using the 021primers. Lane 1=molecular weight marker, lane 2=H27; lane 3=H28; lane4=H29; lane 5=H30; lane 6=molecular weight marker, lane 7=H31; lane8=H32; lane 9=H33; lane 10=H34. Molecular weight marker as in FIG. 1a.

FIG. 5b shows amplification products from 9 faecal samples using the CP1primers. Lane 1=molecular weight marker; lane 2=F1; lane 3=F9; lane4=F10; lane 5=F11; lane 6=F20; lane 7=F21; lane 8=F22; lane 9=F35; lane10=F36; lane 11=F38; lane 12=negative control.

FIGS. 6A-6N illustrate an alignment of Human and Calf sequences of thediagnostic 02 fragment. Figures are to be read in sequential order,i.e., FIGS. 6A to 6N.

EXAMPLES Cryptosporidium Isolates

Isolates of Cryptosporidium are listed in Table 1, below.Cryptosporidium isolates for RAPD analysis were purified from faecal DNAby PBS-ether centrifugation followed by Ficoll-density centrifugation asdescribed by Morgan, Constantine, O'Donoghue, Meloni, O'Brien &Thompson, (1995). "Molecular Characterisation of Cryptosporidiumisolates from humans and other animals using RAPD (Random AmplifiedPolymorphic DNA) analysis. "American Journal of Tropical Medicine andHygiene" 52 559-564. All faecal samples were stored at 4° C. withoutpreservatives for several weeks prior to analysis.

                  TABLE 1                                                         ______________________________________                                        Isolates of Cryptospridium used in this study                                   Code   Host      Species Geographic origin                                                                           Source                               ______________________________________                                        H1   Human     C. parvum Perth, Western Australia                                                                    PMH                                      H2 Human C. parvum Narrogin, SHL                                                 Western Australia                                                          H3 Human C. parvum Nannup, Western Australia PMH                              H4 Human C. parvum Perth, Western Australia PMH                               H5 Human C. parvum Perth, Western Australia PMH                               H6 Human C. parvum Perth, Western Australia PMH                               H7 Human C. parvum Perth, Western Australia PMH                               H8 Human C. parvum Perth, Western Australia SHL                               H9 Human C. parvum Perth, Western Australia SHL                               H10 Human C. parvum Perth, Western Australia PMH                              H11 Human C. parvum Perth, Western Australia SHL                              H12 Human C. parvum Perth, Western Australia SHL                              H13 Human C. parvum Horsham, Victoria CVL                                     H14 Human C. parvum Port Lincon, CVL                                             South Australia                                                            H15 Human C. parvum Perth, Western Australia PMH                              H16 Human C. parvum Perth, Western Australia PMH                              H17 Human C. parvum Perth, Western Australia SHL                              H18 Human C. parvum Perth, Western Australia PMH                              H19 Human C. parvum Perth, Western Australia PMH                              H20 Human C. parvum Perth, Western Australia PMH                              H21 Human C. parvum Perth, Western Australia PMH                              H22 Human C. parvum Perth, Western Australia SHL                              H23 Human C. parvum Perth, Western Australia SHL                              H24 Human C. parvum Perth, Western Australia SHL                              H25 Human C. parvum Perth, Western Australia SHL                              H26 Human C. parvum Perth, Western Australia SHL                              H27 Human C. parvum Perth, Western Australia PMH                              H28 Human C. parvum Perth, Western Australia PMH                              H29 Human C. parvum Bunbury, Western Australia SHL                            H30 Human C. parvum Perth, Western Australia SHL                              H31 Human C. parvum Perth, Western Australia PMH                              H32 Human C. parvum Perth, Western Australia SHL                              H33 Human C. parvum Perth, Western Australia PMH                              H34 Human C. parvum Perth, Western Australia PMH                              F1 Human C. parvum Perth, Western Australia SHL                               F9 Human C. parvum Perth, Western Australia SHL                               F10 Human C. parvum Newman, SHL                                                  Western Australia                                                          F11 Human C. parvum Newman, SHL                                                  Western Australia                                                          F20 Human C. parvum Perth, Western Australia SHL                              F21 Human C. parvum Perth, Western Australia SHL                              F22 Human C. parvum Perth, Western Australia SHL                              F35 Human C. parvum Perth, Western Australia PMH                              F36 Human C. parvum Perth, Western Australia PMH                              F38 Human C. parvum Perth, Western Australia SHL                              C1 Calf C. parvum Millicent, South Australia CVL                              C2 Calf C. parvum Lucindale, South Australia CVL                              C3 Calf C. parvum Meadows, South Australia CVL                                C4 Calf C. parvum Lucindale, South Australia CVL                              C5 Calf C. parvum Penola, South Australia CVL                                 C6 Calf C. parvum Willunga, South Australia CVL                               C7 Calf C. parvum Penola, South Australia CVL                                 C9 Calf C. parvum Maryland, U.S.A. USDA                                       L1 Lamb/Deer C. parvum Edinburgh, Scotland CVL                                S1 Snake C. serpentis Tanunda, South Australia CVL                            S2 Snake C. serpentis Tanunda, South Australia CVL                          ______________________________________                                         (N.B. PMH = Princess Margaret Hospital, Perth, Western Australia; SHL =       State Health Laboratories, Western Australia; CVL = Central Veterinary        Laboratories, Southern Australia Dept. of Agriculture, Southern Australia     ; MAH = Moredun Animal Health Ltd, Edinburgh, Scotland, and USDA = United     States Department of Agriculture, Maryland, U.S.A.; N/A = not available).

DNA Isolation

For RAPD analysis, DNA was extracted from Cryptosporidium using the CTABmethod described by Yap and Thompson (1987). "CTAB precipitation ofcestode DNA". Parasitology Today 3: 220-222. Cryptosporidium oocystswere resuspended in 200 μl of lysis buffer containing, 0.25M Sucrose; 50mM Tris-HCl; 50 mM EDTA; 8% Triton-X-100; pH 7.5. Oocysts were subjectedto three freeze-thaw cycles and then 50 μl of a 10 mg/ml proteinase Ksolution was added. Samples were incubated for 1 hour at 55° C. andnucleic acid precipitated by the addition of 1 ml of 2% CTAB(cetyltrimethylammonium bromide). Following centrifugation, the pelletwas dissolved in 250 μl of N.E. buffer (2.5 M NaCl, 10 mM EDTA, pH 7.7)and diluted with 250 μl of T.E. buffer (10 mM Tris-HCl, 1 mM EDTA, pH7.5). Samples were subsequently chloroform extracted once, precipitatedwith 100% ethanol, washed with 70% ethanol and resuspended in T.E.buffer. DNA was similarly isolated from human blood, human faeces,Giardia duodenalis, Tritrichomonas foetus and C. seprentis forcross-hybridisation studies.

PCR Conditions and Primers

The selection of DNA primer(s) or probe(s) by the construction andscreening of genomic DNA libraries is a laborious and expensiveexercise. However by using the Random Amplified Polymorphic DNA (RAPD)technique described hereafter, for the development of diagnostic probesor primers, the process of selecting such nucleotide sequences isgreatly simplified. In this technique, small amounts of DNA aresubjected to PCR using a single oligonucleotide of random sequence as aprimer. The amplification products are resolved on agarose orpolyacrylamide gels giving rise to a pattern that is strain specific.Many of the products generated by RAPD-PCR are derived from repetitiveDNA sequences. As these sequences are frequently species-specific,RAPD-PCR is potentially a quick method for developing species-specificdiagnostic PCR primers and probes

RAPD reactions were performed as described by Morgan, Constantine,O'Donoghue, Meloni, O'Brien & Thompson, (1995). "MolecularCharacterisation of Cryptosporidium isolates from humans and otheranimals using RAPD (Random Amplified Polymorphic DNA) analysis."American Journal of Tropical Medicine and Hygiene" 52 559-564. A rangeof primers were tested and are listed below.

    R-2817      5' GCTTGGTCTGCTCAATGTGG 3'                                           - INS     5' ACAGGGGTGTGGGG 3'                                                - PER     5' GACNGGNACNGG 3'                                                  - Y22     5' CTCTGGGTGTCGTGC 3'                                               - SP6     5' GATTTAGGTGACACTATAG 3'                                           - [GAA].sup.5  5' GAAGAAGAAGAAGAA 3'                                          - [GACA].sup.4 5' GACAGACAGACAGACA 3'                                         - R4      5' AGTCGAACCCTGATTCTCCGCCAGG 3'                              

Vacuum Blots, Dot Blots and DNA Hybridisation

RAPD gels were vacuum blotted (BioRad) onto Hybond N⁺ ([Amersham)membranes using 20×SSC (0.3 M Na₃ citrate; 3 M NaCl; pH adjusted to 7.0)as the transfer medium. Following transfer, DNA was UV cross-linked tothe membranes using a GS Gene-Linker™ UV cross-linker (BioRad). Probelabelling was conducted using two different non-radioactive labellingsystems. The ECL (Enhanced Chemiluminescence) direct labelling kitsupplied by Amersham, was used to label all double stranded DNA and theDIG (digoxigenin) oligonucleotide 3'-end labelling and detection kitsupplied by Boehringer Mannheim was used to label oligonucleotides. Formost hybridisations, 100 ng of DNA (at a concentration of 10 ng/μl) waslabelled and used in a 10 ml hybridisation volume. All hybridisationswere carried out in a Hybaid™ rotisserie oven (BioRad). For dot-blots,DNA was transferred to Hybond N⁺ membrane (Amersham), using a vacuummanifold (BioRad). DNA was bound to the membrane using the UVcross-linking procedure described above.

Southern blots of RAPD profiles were hybridised to DNA isolated fromhuman blood, Giardia duodenalis, human faeces and Cryptosporidiumparvum. RAPD bands which hybridised only to Cryptosporidium DNA and notto the other DNA's tested were chosen for further analysis. PrimersR-2817, INS, PER, Y22, SP6, [GAA]⁵ and [GACA]⁴ all produced profileswhich cross-reacted to varying degrees with human, faecal or GiardiaDNA. The primer R4, however, produced a simple profile whichcross-reacted with Cryptosporidium DNA only (data not shown). A band ofapproximately 750 bp was purified from a low-melting point gel using thesyringe method described by Li. & Ownby (1993). "A rapid method forextraction of DNA from agarose gels using a syringe." Biotechniques 15:976-978, reamplified and shown to be specific for Cryptosporidium bydot-blots. The band, designated 021 was then cloned and sequenced.

Cloning of PCR Products

Bands specific for Cryptosporidium were ligated directly into the pGEMT-Vector (Promega). Ligation products were transformed into Escherichiacoli HB101 and white colonies were screened using PCR. Half of eachwhite colony was removed with a sterile toothpick and added to a 50 μlsolution of TE buffer containing 1% Triton-X-100. The toothpick wasswirled around to dislodge the cells and then discarded. These tubeswere subsequently incubated at 95° C. for 10 min to lyse the cells, spunfor 5 min to remove cellular debris and the supernatant transferred to aclean tube.

A 5 μl aliquot of this supernatant was used in a PCR reaction, with theM13 foreword and reverse primers. Briefly, 5 μl of crude lysate wasamplified in 67 mM Tris-HCL (pH 7.6), 16.6 mM (NH₄)₂ SO₄ ; 2 mM MgCl₂ ;200 μM of each dNTP; 12.5 pmoles of each primer; 0.5 units of Tth Plus(Biotech International) and sterile distilled water. Reactions wereperformed on an OmniGene thermal cycler (Hybaid), using the followingcycling conditions. One cycle of 94° C. for 2 min; 55° C. for 2 min and72° C. for 2 min, followed by 30 cycles of 94° C. for 30 seconds; 55° C.for 1 min and 72° C. for 2 min with a final cycle of 94° C. for 30seconds; 55° C. for 1 min and 72° C. for 10 min. An aliquot (5-10 μl) ofthe amplified product was then run on a 1% agarose gel and checked forsize.

At least 10 white colonies for each ligation were checked for thepresence of inserts using the PCR protocol described above. Inserts werecut out using Sac II and Pst 1 restriction enzymes (Pharmacia),electrophoresed on a 1% low-melting point agarose gel and the insertpurified from the gel using the syringe method. At this point, insertswere again checked for specificity by dot blots andCryptosporidium-specific inserts were chosen for sequencing.

Sequencing and Synthesis of Primers

Sequencing was carried out using the Taq DyeDeoxy™ Terminator CycleSequencing Kit supplied by Applied Biosystems. Sequences were alignedusing the Seqed and DNA strider programs and compared with Genebank andEMBL databases for sequence homology. A number of primer sequences weredesigned from the 021 sequence using the computor program Amplify™ andoligonucleotides were synthesised by DNA Express.

Primers

The 021 forward and 021 reverse primers which produced a 668 bp fragmentupon amplification of Cryptosporidium parvum DNA are listed below. Anoligonucleotide internal to the sequence amplified by the 021 primerswas also synthesised for use as a probe to confirm parasite origin ofthe amplified product. A second set of primers, designated CP1 forwardand reverse and an internal oligonucleotide designated CPI were alsodesigned from the 021 sequence. These primers produced an approximately426 bp fragment upon amplification of Cryptosporidium DNA.

    021 F        5' GGTACTGGATAGATAGTGGA 3'                                          - 021 R  5' TCGCACGCCCGGATTCTGTA 3'                                           - Oligo  5' AGTCCCGTATCAGTTCGAGA 3'                                           - CP1 F  5' GAGATTCTGAAATTAATTGG 3'                                           - CP1 R  5' CCTCCTTCGTTAGTTGAATCC 3'                                          - CPI    5' GTTGGCTTATAGATTCTGAGC 3'                                   

The sequence of the diagnostic fragment is shown below with thepositions at which the primers bind underlined. The CP1 forward andreverse primers are shown binding inside the sequence specified by the021 primers and produce a 426 bp product upon amplification.

    GATGGTACTGGATAGATAGTGGAAGTCCCGTATCAGTTCGAGATTCTGAAATTAATTGG                      - ACATCAAGTTATAAAGCAAGCTGGTTATTAAGATTCAAATTTCCCTTTGAAAAGTGTGG                 - CTTTTTTGATATTGGAGGGTTAGGAAGAAGGCCGTGTTGGCTTATAGATTCTGAGCTTT                 - CTTGTGCAGTTTGTGGTACAGTAGCTTATGATTCTGGTGGGCTGAATCCCAATAAATAT                 - TCAGAGCTAATTAAGCAGACTGATGAAATTATTAGTAAAGAGCCAAAGCTTGATCTTCC                 - AGGTTACAATAATTTGAACTGTACAGATGCTTGGGAGAATAATTTATCAGTTGGTCTTT                 - GTCAAAATGTCTCAAATATCCTGGACTCAGCTTGGAGCTCGTATCAGAGTTCGTTAAAC                 - TTTCCTAGTATCAACTTTAACTGGAAAGAGGATTCAACTAACGAAGGAGGGGACCAAGT                 - TTACCATAATTCTTATTTGGATCTTCCAAGGTATAAGCAGAAGAAAACATTTTATTGGG                 - ATCAGGATCCAGGTACTATTCCAGCTTTGTCTGATGAAATGAAGCTCATTGGTTTAAGC                 - GCTCAACCAACATACCATCCTTTGGATAGAAGCTCATCTGGAAGTTTTGAGTCTGATAG                 - TACAGAATCCGGGCGTGCGAATGAAGAAAGAAACGATAC                              

Diagnostic PCR Conditions

PCR conditions for the 021 diagnostic PCR primers consisted of 67 mMTris-HCL (pH 7.6), 16.6 mM (NH₄)₂ SO₄ ; 1.5 mM MgCl₂ ; 200 μM of eachdNTP; 6.5 pmoles of each primer; 0.25 units of Tth Plus (BiotechInternational) and sterile distilled water. Reactions were performed onan OmniGene thermal cycler (Hybaid), using the following cyclingconditions. One cycle of 94° C. for 2 min; 58° C. for 2 min and 72° C.for 2 min, followed by 40 cycles of 94° C. for 30 seconds; 58° C. for 1min and 72° C. for 2 min with a final cycle of 94° C. for 30 seconds;58° C. for 1 min and 72° C. for 10 min. PCR conditions for the CP1primers were essentially the same except that 2 mM MgCl₂ and anannealing temperature of 59° C. was used.

Diagnostic Test

For sensitivity testing, crude oocyst preparations were resuspended in10 μl of T.E. Decreasing concentrations of oocyst suspensions wereprepared by serial dilutions. For direct PCR analysis of faecal samples,0.5 g of faeces was mixed with 4 ml PBS and this slurry was then diluted1 in 20 in T.E. Samples were then freeze-thawed 3 times, boiled for 5min, spun for 1 min to remove debris and then 5-10 μl of the supernatantwas added directly to the PCR reaction.

The above oligonucleotide sequences are unique in that a comparison ofthe sequence information obtained from the 021 clone with Genebank andEMBL databases produced no homology of any significance. The specificityof the primers designed from the 021 clone was tested by performing PCRreactions on DNA extracted from Giardia duodenalis, human blood, humanfaeces, Tritrichomonas foetus, and C. serpentis. With both sets ofprimers, DNA of the correct size was amplified from Cryptosporidiumparvum DNA only. No amplification was seen with any of the other DNA'stested (see FIGS. 1a and b).

The primers were also tested on Cryptosporidium of both human and bovineorigin and the PCR products confirmed by hybridisation to the internaloligonucleotide. These diagnostic primers were then used to amplify over40 different isolates of Cryptosporidium parvum of both human and bovineorigin (listed in Table 1), to determine if the primers would recognisesome or all isolates. All isolates tested produced the correct sizedupon amplification (see FIGS. 2a; 2b and 3).

The gel depicted in FIG. 2a was then blotted onto Hybond N⁺ (Amersham)and probed with the internal oligonuleotide probes to confirm parasiteorigin of bands (FIG. 2b).

The amplification products of the CPF primers were also probed with aninternal oligonucleotide to confirm parasite origin of the bands. In allcases the 446 bp amplification product hybridised strongly with theinternal olifo indicating that the reaction was specific forCryptosporidium (data not shown).

The detection limits of the primers were found to be as high as oneoocyst (see FIG. 4a) with both the 021 and the CP1 primers (see FIG. 4b)when amplifying from crude preparations of oocysts.

The primers were also used to reproducibly amplify Cryptosporidiumdirectly from boiled faeces (see FIG. 5a). Most of the eight faecalsamples tested contained relatively low numbers of oocysts (ranging from1×10³ to 5×10⁵ oocysts per gram of faeces, with one sample, H29,containing 1.5×10⁶ oocysts per g of faeces). One sample, H27, unlike theother samples, was a solid stool and it was necessary to perform a crudePBS-ether extraction of that sample in order to obtain a reproducibleamplification product (see FIG. 5a).

FIG. 5b shows amplification products from 9 faecal samples using the CP1primers. Lane 1=molecular weight marker; lane 2=F1; lane 3=F9; lane4=F10; lane 5=F11; lane 6=F20; lane 7=F21; lane 8=F22; lane 9=F35; lane10=F36; lane 11=F38; lane 12=negative control.

The sequences of two human and two calf isolates of Cryptosporidiumparvum were compared along the length of the diagnostic fragment todetermine the extent of sequence conservation between isolates (seeFIGS. 6A-6N). Direct PCR sequencing was carried out using the TaqDyeDeoxy™ Terminator Cycle Sequencing Kit supplied by AppliedBiosystems. Sequences were aligned using the CLUSTAL V multiple sequencealignment program. The alignment shows the sequence to be conservedbetween isolates but with a number of sequence differences between thehuman and calf isolates. These findings are in keeping with RAPDanalysis on these isolates described by Morgan, Constantine, O'Donoghue,Meloni, O'Brien & Thompson, (1995). "Molecular Characterisation ofCryptosporidium isolates from humans and other animals using RAPD(Random Amplified Polymorphic DNA) analysis. "American Journal ofTropical Medicine and Hygiene" 52 559-564, which reported geneticdifferences between human and calf isolates. The observed differencesbetween the human and calf isolates is not sufficient to interfere withprimer binding and both human and calf isolates are amplified using boththe 021 and the CP1 primers (primer sequences are underlined). Given thedifferences between the human and calf isolates however, it would bepossible to construct primers which could differentiate between humanand animal isolates (ie a set of primers which would amplify humanisolates only and a second set which would amplify calf/animal isolatesonly). Primers which are specific for animal or human isolates would bevery useful in transmission studies and also for environmental analysisin determining the likely source of contamination of water supplies (iehuman or animal).

The above mentioned sequence was analysed to determine whether or notthe sequence was from a coding or non-coding section of DNA. A number ofcomputer programs were used including CODON PREFERENCE which is aframe-specific gene finder that tries to recognise protein codingsequences by virtue of the similarity of their codon usage to a codonfrequency table or by the bias of their composition (usually GC) in thethird position of each codon. Analysis of the sequence using theseprograms however, suggests that the 021 fragment is unlikely to containcoding regions.

RAPD analysis was used to develop diagnostic primers for Cryptosporidiumparvum which have been shown to be both specific and also sensitive.Both the 021 and the CP1 primers appear to be very specific forCryptosporidium, can detect as little as one oocyst and can amplifyCryptosporidium directly from boiled faeces. Over 47 different isolatesof Cryptosporidium parvum of both human and bovine origin from diversegeographic locations were screened using these primers and all amplifiedthe correct sized band, indicating that the sequence defined by theprimers is conserved amongst isolates.

The specificity testing of the RAPD primers did not includeCryptosporidium baileyi, Cryptosporidium meleagridis or Cryptosporidiummuris DNA, as a source of this material was not readily available.Although Cryptosporidium muris has been reported in cattle in the UnitedStates, and oocysts resembling Cryptosporidium baileyi were recoveredfrom an immunocompromised human patient, these species ofCryptosporidium are not commonly reported in livestock and particularlynot from humans. Further optimisation of the assay is required to enablethe amplification of all Cryptosporidium isolates directly from faeceswithout any prior purification.

The RAPD primers described herein could be used both in the diagnosis ofCryptosporidium from faecal samples and also in environmentalmonitoring. The level of skill required by microscopic identification ofCryptosporidium oocysts, the low sensitivity of current diagnosticmethods and varying expertise among laboratories and technicians canresult in many cases of mild cryptosporidial infection remainingundiagnosed. A simple PCR detection system employing primers of thepresent invention should greatly improve the detection and diagnosis ofCryptosporidium.

Outbreaks of cryptosporidiosis in child day care centres are frequentlyreported (Alpert et al. 1986; Crawford et al. 1988; Diers & McCallister1989; Ferson & Young 1992; Hanna & Brooks 1995), and family members areoften affected during such outbreaks. A proportion of each group isasymptomatic and can act as carriers of infection to relatives and thecommunity. Therefore, the public health problem of transmission to thecommunity from these centres is a significant one and needs furtherevaluation and control. Investigations undertaken during outbreaks ofdiarrhoea however, have frequently used limited diagnostic testing thathave tended to incriminate agents that are easily identifiable instandard microbiological laboratories (Thompson 1994). Sensitivemolecular-based tools such as the PCR primers described here will allowmore accurate molecular-epidemiological studies to be carried out todetermine not only the true prevalence of Cryptosporidium in thecommunity, but also the risk factors associated with infection.

A recent survey in the United States conducted by Clancy et al. (1994)"Commercial labs: how accurate are they?" Journal of the American WaterWorks Association. 86: 89-97, revealed that commercial laboratoriesshowed a lack of proficiency in testing water samples for Giardia andCryptosporidium. With the implementation in 1995 of the InformationCollection Rule (ICR) in the United States which makes testing forGiardia and Cryptosporidium in water systems serving more than 10,000people mandatory, the development of accurate and sensitive testing forCryptosporidium is of great importance.

As a result of this study, highly sensitive and specific diagnostic PCRprimers have been developed for Cryptosporidium.

REFERENCES

ALPERT, G. L. M., BELL, C. E., KIRKPATRICK L. D., BUDNICK, J. M.,CAMPOS, H. M., FRIEDMAN, H. M. AND PLOTKIN, S. A. (1986) Outbreak ofcryptosporidiosis in a day-care centre. Pediatrics. 77, 152-156.

CRAWFORD, F. G., VERMUND S. H., MA, J. Y. AND DECKELBAUM, R. J. (1988).Asymptomatic cryptosporidiosis in a New York City day care centre.Paediatric Infectious Disease Journal. 7, 806.

DIERS, J. & McCALLISTER G. L. (1989). Occurrence of Cryptosporidium inhome daycare centres in West-Central Colorado. Journal of Parasitology.75, 637-638.

FERSON, M. J. & YOUNG, L. C. (1992). Cryptosporidium and coxsackievirusB5 causing epidemic diarrhoea in a child-care centre. Medical Journal ofAustralia. 156, 813.

HANNA J. & BROOKS, D. (1995). Cryptosporidiosis in a child day-carecentre. Communicable Disease Intelligence. 19, 6-7.

THOMPSON, S. C. (1994). Infectious diarrhoea in children--controllingtransmission in the child care setting. Journal of Paediatric ChildHealth. 30, 210-219.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 68                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 688 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GATGGTACTG GATAGATAGT GGAAGTCCCG TATCAGTTCG AGATTCTGAA AT -            #TAATTGGA     60                                                                 - - CATCAAGTTA TAAAGCAAGC TGGTTATTAA GATTCAAATT TCCCTTTGAA AA -            #GTGTGGCT    120                                                                 - - TTTTTGATAT TGGAGGGTTA GGAAGAAGGC CGTGTTGGCT TATAGATTCT GA -            #GCTTTCTT    180                                                                 - - GTGCAGTTTG TGGTACAGTA GCTTATGATT CTGGTGGGCT GAATCCCAAT AA -            #ATATTCAG    240                                                                 - - AGCTAATTAA GCAGACTGAT GAAATTATTA GTAAAGAGCC AAAGCTTGAT CT -            #TCCAGGTT    300                                                                 - - ACAATAATTT GAACTGTACA GATGCTTGGG AGAATAATTT ATCAGTTGGT CT -            #TTGTCAAA    360                                                                 - - ATGTCTCAAA TATCCTGGAC TCAGCTTGGA GCTCGTATCA GAGTTCGTTA AA -            #CTTTCCTA    420                                                                 - - GTATCAACTT TAACTGGAAA GAGGATTCAA CTAACGAAGG AGGGGACCAA GT -            #TTACCATA    480                                                                 - - ATTCTTATTT GGATCTTCCA AGGTATAAGC AGAAGAAAAC ATTTTATTGG GA -            #TCAGGATC    540                                                                 - - CAGGTACTAT TCCAGCTTTG TCTGATGAAA TGAAGCTCAT TGGTTTAAGC GC -            #TCAACCAA    600                                                                 - - CATACCATCC TTTGGATAGA AGCTCATCTG GAAGTTTTGA GTCTGATAGT AC -            #AGAATCCG    660                                                                 - - GGCGTGCGAA TGAAGAAAGA AACGATAC         - #                  - #                688                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GGTACTGGAT AGATAGTGGA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TCGCACGCCC GGATTCTGTA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - AGTCCCGTAT CAGTTCGAGA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - ACTGGATAGA TAGTGGAAGT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - TTTCTTTCTT CATTCGCACG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GTGGAAGTCC CGTATCAGTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - ACGCCCGGAT TCTGTACTAT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GATAGATAGT GGAAGTCCCG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - CTGAAATTAA TTGGACATCA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GTACTATCAG ACTCAAAACT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GTGGTACTGG ATAGATAGTG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GTATCGTTTC TTTCTTCATT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - TGGTACTGGA TAGATAGTGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - TATCGTTTCT TTCTTCATTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - TAGATAGTGG AAGTCCCGTA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - TCTTCATTCG CACGCCCGGA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - ATAGTGGAAG TCCCGTATCA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - TTTCTTCATT CGCACGCCCG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - CTGGATAGAT AGTGGAAGTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - CGTTTCTTTC TTCATTCGCA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - TAATTGGACA TCAAGTATAA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - TCTGAAATTA ATTGGACATC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - CTTCCAGATG AGCTTCTATC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - GGTGGTACTG GATAGATAGT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - GGTATCGTTT CTTTCTTCAT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - GAGATTCTGA AATTAATTGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - GTTGGCTTAT AGATTCTGAG C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - GGTTATTAAG ATTCAAATTT CC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - TCCCGTATCA GTTCGAGATT CTG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - CGAACTCTGA TACGAGCTCC AAGC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - ATTCGAGATT CTGAAATTAA TTGG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - GAATAGTACC TGGATCCTGA TCCC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - GATATTGGAG GGTTAGGAAG AAGG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - CTGTACAGTT CAAATTATTG TAACC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - GACTGATGAA ATTATTAGTA AAGAGC          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - CCTCCTTCGT TAGTTGAATC CTC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - CAGTTCAAAT TATTGTAGCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - GTTCGAGATT CTGAAATTAA TTGG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - GTCCCGTATC AGTTCGAGAT TCTG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - GGAGGGTTAG GAAGAAGGCC GTG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - GCTTGGGAGA ATAATTTATC AG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - GGGATCAGGA TCCAGGTACT ATTC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - GTATCGTTTC TTTCTTCATT CGC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - GGACCAAGTT TACCATAATT C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - GGAGAATAAT TTATCAGTTG GTC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - CAAGGTATAA GCAGAAGAAA AC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - CGCACGCCCG GATTCTGTAC TATC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - ATGTCTCAAA TATCCTGGAC TCAG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - GTACTGGATA GATAGTGGAA GTC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - CACGGCCTTC TTCCTAACCC TCC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - GGAAGTCCCG TATCAGTTCG AG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - CCTCCTTCGT TAGTTGAATC C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - CAGTTCAAAT TATTGTAACC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - TAATTGGACA TCAAGTTATA AAGC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - GGAAGATCCA AATAAGAATT ATGG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - GGTACTGGAT AGATAGTGGA AG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - CCAGAATCAT AAGCTACTGT ACC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - CCTCCTTCGT TAGTTGAATC C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - CAGTTCAAAT TATTGTAACC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - GCTTGGTCTG CTCAATGTGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - ACAGGGGTGT GGGG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - GACNGGNACN GG              - #                  - #                      - #       12                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - CTCTGGGTGT CGTGC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - GATTTAGGTG ACACTATAG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - - GAAGAAGAAG AAGAA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - - GACAGACAGA CAGACA             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - - AGTCGAACCC TGATTCTCCG CCAGG          - #                  - #                   25                                                                    __________________________________________________________________________

We claim:
 1. A purified and isolated Cryptosporidium DNA sequencecomprising the nucleotide sequence:

    GATGGTACTGGATAGATAGTGGAAGTCCCGTATCAGTTCGAGATTCTGAAA                             - TTAATTGGACATCAAGTTATAAAGCAAGCTGGTTATTAAGATTCAAATTTC                         - CCTTTGAAAAGTGTGGCTTTTTTGATATTGGAGGGTTAGGAAGAAGGCCGT                         - GTTGGCTTATAGATTCTGAGCTTTCTTGTGCAGTTTGTGGTACAGTAGCTT                         - ATGATTCTGGTGGGCTGAATCCCAATAAATATTCAGAGCTAATTAAGCAGA                         - CTGATGAAATTATTAGTAAAGAGCCAAAGCTTGATCTTCCAGGATACAATA                         - ATTTGAACTGTACAGATGCTTGGGAGAATAATTTATCAGTTGGTCTTTGTC                         - AAAATGTCTCAAATATCCTGGACTCAGCTTGGAGCTGGTATCAGAGTTCGT                         - TAAACTTTCCTAGTATCAACTTTAACTGGAAAGAGGATTCAACTAACGAAG                         - GAGGGGACCAAGTTTACCATAATTCTTATTTGGATCTTCCAAGGTATAAGC                         - AGAAGAAAACATTTTATTGGGATCAGGATCCAGGTACTATTCCAGCTTTGT                         - CTGATGAAATGAAGCTCATTGGTTTAAGCGCTCAACCAACATACCATCCTT                         - TGGATAGAAGCTCATCTGGAAGTTTTGAGTCTGATAGTACAGAATCCGGGC                         - GTGCGAATGAAGAAAGAAACGATAC (SEQ ID NO. 1).                             


2. A method for detecting and/or identifying the presence ofCryptosporidium genomic material in a sample, said method comprising thesteps of:(i) selecting at least a primer(s) or probe from nucleotidesequence according to claim 1; and (ii) using the primer(s) or probe todetect and/or identify the presence of Cryptosporidium genomic materialin the sample.
 3. A method according to claim 2 wherein Cryptosporidiumgenomic material in the sample is detected by a hybridisation assay. 4.A method according to claim 2 wherein the probe or primer(s) is at least5 nucleotides in length.
 5. A method according to claim 2 wherein theprobe or primer(s) is about 10 to 50 nucleotides in length.
 6. A methodaccording to claim 2 wherein the probe or primer(s) is about 20 to 24nucleotides in length.
 7. A method according to claim 3 wherein theprobe or primer(s) is selected from from the group of sequencesconsisting of:

    GGTACTGGATAGATAGTGGA;                                      (SEQ ID NO. 2);

    TCGCACGCCCGGATTCTGTA;                                      (SEQ ID NO. 3);

    AGTCCCGTATCAGTTCGAGA;                                      (SEQ ID NO. 4);

    ACTGGATAGATAGTGGAAGT;                                      (SEQ ID NO. 5);

    TTTCTTTCTTCATTCGCACG;                                      (SEQ ID NO. 6);

    GTGGAAGTCCCGTATCAGTC;                                      (SEQ ID NO. 7);

    ACGCCCGGATTCTGTACTAT;                                      (SEQ ID NO. 8);

    GATAGATAGTGGAAGTCCCG;                                      (SEQ ID NO. 9);

    ACGCCCGGATTCTGTACTAT;                                      (SEQ ID NO. 8);

    CTGAAATTAATTGGACATCA;                                      (SEQ ID NO. 10);

    GTACTATCAGACTCAAAACT;                                      (SEQ ID NO. 11);

    GTGGTACTGGATAGATAGTG;                                      (SEQ ID NO. 12);

    GTATCGTTTCTTTCTTCATT;                                      (SEQ ID NO. 13);

    TGGTACTGGATAGATAGTGG;                                      (SEQ ID NO. 14);

    TATCGTTTCTTTCTTCATTC;                                      (SEQ ID No. 15);

    TAGATAGTGGAAGTCCCGTA;                                      (SEQ ID NO. 16);

    TCTTCATTCGCACGCCCGGA;                                      (SEQ ID NO. 17);

    ATAGTGGAAGTCCCGTATCA;                                      (SEQ ID NO. 18);

    TTTCTTCATTCGCACGCCCG;                                      (SEQ ID NO. 19);

    CTGGATAGATAGTGGAAGTC;                                      (SEQ ID NO. 20);

    CGTTTCTTTCTTCATTCGCA;                                      (SEQ ID NO. 21);

    TAATTGGACATCAAGTATAA;                                      (SEQ ID NO. 22);

    GTACTATCAGACTCAAAACT;                                      (SEQ ID NO. 11);

    TCTGAAATTAATTGGACATC;                                      (SEQ ID NO. 23);

    CTTCCAGATGAGCTTCTATC;                                      (SEQ ID NO. 24);

    GGTGGTACTGGATAGATAGT;                                      (SEQ ID NO. 25);

    GGTATCGTTTCTTTCTTCAT;                                      (SEQ ID NO. 26);

    GAGATTCTGAAATTAATTGG;                                      (SEQ ID NO. 27);

    GTTGGCTTATAGATTCTGAGC;                                     (SEQ ID NO. 28);

    GGTTATTAAGATTCAAATTTCC;                                    (SEQ ID NO. 29);

    TCCCGTATCAGTTCGAGATTCTG;                                   (SEQ ID NO. 30);

    CGAACTCTGATACGAGCTCCAAGC;                                  (SEQ ID NO. 31);

    ATTCGAGATTCTGAAATTAATTGG;                                  (SEQ ID NO. 32);

    GAATAGTACCTGGATCCTGATCCC;                                  (SEQ ID NO. 33);

    GATATTGGAGGGTTAGGAAGAAGG;                                  (SEQ ID NO. 34);

    CTGTACAGTTCAAATTATTGTAACC;                                 (SEQ ID NO. 35);

    GACTGATGAAATTATTAGTAAAGAGC;                                (SEQ ID NO. 36);

    CCTCCTTCGTTAGTTGAATCCTC;                                   (SEQ ID NO. 37);

    TCGCACGCCCGGATTCTGTA;                                      (SEQ ID NO. 3);

    CAGTTCAAATTATTGTAGCC;                                      (SEQ ID NO. 38);

    GTTCGAGATTCTGAAATTAATTGG;                                  (SEQ ID NO. 39);

    GTCCCGTATCAGTTCGAGATTCTG;                                  (SEQ ID NO. 40);

    GGAGGGTTAGGAAGAAGGCCGTG;                                   (SEQ ID NO. 41);

    GCTTGGGAGAATAATTTATCAG;                                    (SEQ ID NO. 42);

    GGGATCAGGATCCAGGTACTATTC;                                  (SEQ ID NO. 43);

    GTATCGTTTCTTTCTTCATTCGC;                                   (SEQ ID NO.44);

    GGACCAAGTTTACCATAATTC;                                     (SEQ ID NO. 45);

    GGAGAATAATTTATCAGTTGGTC;                                   (SEQ ID NO. 46);

    CAAGGTATAAGCAGAAGAAAAC;                                    (SEQ ID NO. 47);

    CGCACGCCCGGATTCTGTACTATC;                                  (SEQ ID NO. 48);

    ATGTCTCAAATATCCTGGACTCAG;                                  (SEQ ID NO. 49);

    GTACTGGATAGATAGTGGAAGTC;                                   (SEQ ID NO. 50);

    CACGGCCTTCTTCCTAACCCTCC;                                   (SEQ ID NO. 51);

and

    GGAAGTCCCGTATCAGTTCGAG                                     (SEQ ID NO. 52).


8. 8. A method for detecting and/or identifying microorganisms of thegenus Cryptosporidium, comprising the steps of:(i) selecting at least aset of primers from the nucleotide sequence defined in claim 1 which arespecific for Cryptosporidium DNA; (ii) mixing the primers with a samplesuspected of containing Cryptosporidium DNA; (iii) amplifying theproduct(s) of step (ii) by the polymerase chain reaction; and (iv)detecting the presence of the product of step (iii).
 9. A methodaccording to claim 7 wherein the primers are selected from the group ofprimer pairs consisting of:

    (i)   5' ACTGGATAGATAGTGGAAGT 3'                                                                        (SEQ ID NO. 5);                                        -     5' TTTCTTTCTTCATTCGCACG 3' (SEQ ID NO. 6);                              - (ii) 5' GTGGAAGTCCCGTATCAGTC 3' (SEQ ID NO. 7);                             -     5' ACGCCCGGATTCTGTACTAT 3' (SEQ ID NO. 8);                              - (iii) 5' GATAGATAGTGGAAGTCCCG 3' (SEQ ID NO. 9);                            -     5' ACGCCCGGATTCTGTACTAT 3' (SEQ ID NO. 8);                              - (iv)5' CTGAAATTAATTGGACATCA 3' (SEQ ID NO. 10);                             -     5' GTACTATCAGACTCAAAACT 3' (SEQ ID NO. 11);                             - (v) 5' GTGGTACTGGATAGATAGTG 3' (SEQ ID NO. 12);                             -     5' GTATCGTTTCTTTCTTCATT 3' (SEQ ID NO. 13);                             - (vi) 5' TGGTACTGGATAGATAGTGG 3' (SEQ ID NO. 14);                            -      5' TATCGTTTCTTTCTTCATTC 3' (SEQ ID NO. 15);                            - (vii) 5' TAGATAGTGGAAGTCCCGTA 3' (SEQ ID NO. 16);                           -       5' TCTTCATTCGCACGCCCGGA 3' (SEQ ID NO. 17);                           - (viii) 5' ATAGTGGAAGTCCCGTATCA 3' (SEQ ID NO. 18);                          -        5' TTTCTTCATTCGCACGCCCG 3' (SEQ ID NO. 19);                          - (ix)   5' CTGGATAGATAGTGGAAGTC 3' (SEQ ID NO. 20);                          -        5' CGTTTCTTTCTTCATTCGCA 3' (SEQ ID NO. 21);                          - (x)    5' TAATTGGACATCAAGTATAA 3' (SEQ ID NO. 22);                          -        5' GTACTATCAGACTCAAAACT 3' (SEQ ID NO. 11);                          - (xi)   5' TCTGAAATTAATTGGACATC 3' (SEQ ID NO. 23);                          -        5' CTTCCAGATGAGCTTCTATC 3' (SEQ ID NO. 24);                          - (xii)  5' GGTGGTACTGGATAGATAGT 3' (SEQ ID NO. 25);                          -        5' GGTATCGTTTCTTTCTTCAT 3' (SEQ ID NO. 26);                          - (xiii) 5' GGTACTGGATAGATAGTGGA 3' (SEQ ID NO. 2);                           -        5' TCGCACGCCCGGATTCTGTA 3' (SEQ ID NO. 3);                           - (xiv)  5' GAGATTCTGAAATTAATTGG 3' (SEQ ID NO. 27);                          -        5' CCTCCTTCGTTAGTTGAATCC 3' (SEQ ID NO. 53);                         - (xv)   5' GTTGGCTTATAGATTCTGAGC 3' (SEQ ID NO. 28);                         -        5' CAGTTCAAATTATTGTAGCC 3' (SEQ ID NO. 38);                          - (xvi)  5' GAGATTCTGAAATTAATTGG 3' (SEQ ID NO. 27);                          -        5' CAGTTCAAATTATTGTAACC 3' (SEQ ID NO. 54);                          - (xvii)   5' GTTGGCTTATAGATTCTGAGC 3' (SEQ ID NO. 28);                       -        5' CCTCCTTCGTTAGTTGAATCC 3' (SEQ ID NO. 53);                         - (xviii) 5' TAATTGGACATCAAGTTATAAAGC 3' (SEQ ID NO. 55);                     -         5' GGAAGATCCAAATAAGAATTATGG 3' (SEQ ID NO. 56);                     - (xix)   5' GGTTATTAAGATTCAAATTTCC 3' (SEQ ID NO. 29);                       -         5' CGAACTCTGATACGAGCTCCAAGC 3' (SEQ ID NO. 31);                     - (xx)    5' TCCCGTATCAGTTCGAGATTCTG 3' (SEQ ID NO. 30);                      -         5' CGAACTCTGATACGAGCTCCAAGC 3' (SEQ ID NO. 31);                     - (xxi)   5' GTTCGAGATTCTGAAATTAATTGG 3' (SEQ ID NO. 39);                     -         5' CGAACTCTGATACGAGCTCCAAGC 3' (SEQ ID NO. 31);                     - (xxii) 5' TAATTGGACATCAAGTTATAAAGC 3' (SEQ ID NO. 55);                      -         5' CGAACTCTGATACGAGCTCCAAGC 3' (SEQ ID NO. 31);                     - (xxiii)   5' GGTTATTAAGATTCAAATTTCC 3' (SEQ ID NO. 29);                     -           5' CGAACTCTGATACGAGCTCCAAGC 3' (SEQ ID NO. 34);                   - (xxiv)    5' TCCCGTATCAGTTCGAGATTCTG 3' (SEQ ID NO. 30);                    -           5' GAATAGTACCTGGATCCTGATCCC 3'  (SEQ ID NO. 33);                  - (xxv) 5' TAATTGGACATCAAGTTATAAAGC 3' (SEQ ID NO. 55);                       -           5' GAATAGTACCTGGATCCTGATCCC 3'  (SEQ ID NO. 33)                   - (xxvi)   5' GGTTATTAAGATTCAAATTTCC 3' (SEQ ID NO. 29);                      -           5' GAATAGTACCTGGATCCTGATCCC 3'  (SEQ ID NO. 33);                  - (xxvii)    5' TCCCGTATCAGTTCGAGATTCTG 3' (SEQ ID NO. 30);                   -         5' GGAAGATCCAAATAAGAATTATGG 3' (SEQ ID NO. 56);                     - (xxviii) 5' GTTCGAGATTCTGAAATTAATTGG 3' (SEQ ID NO. 39);                    -         5' GGAAGATCCAAATAAGAATTATGG 3' (SEQ ID NO. 56);                     - (xxix)   5' GGTTATTAAGATTCAAATTTCC 3' (SEQ ID NO. 29);                      -         5' GGAAGATCCAAATAAGAATTATGG 3' (SEQ ID NO. 56);                     - (xxx)   5' ATTCGAGATTCTGAAATTAATTGG 3'  (SEQ ID NO. 32);                    -           5' GAATAGTACCTGGATCCTGATCCC 3'  (SEQ ID NO. 33);                  - (xxxi)    5' GATATTGGAGGGTTAGGAAGAAGG 3' (SEQ ID NO. 34);                   -         5' CTGTACAGTTCAAATTATTGTAACC 3'  (SEQ ID NO. 35);                   - (xxxii) 5' GACTGATGAAATTATTAGTAAAGAGC 3' (SEQ ID NO. 36);                   -         5' CCTCCTTCGTTAGGTGAATCCTC 3' (SEQ ID NO. 37);                      - (xxxiii) 5' GGTACTGGATAGATAGTGGAAG 3' (SEQ ID NO. 57);                      -          5' CCAGAATCATAAGCTACTGTACC 3' (SEQ ID NO. 58);                     - (xxxiv)   5' GTCCCGTATCAGTTCGAGATTCTG 3'  (SEQ ID NO. 40);                  -          5' CCTCCTTCGTTAGTTGAATCCTC 3' (SEQ ID NO. 37);                     - (xxxv)   5' GGGATCAGGATCCAGGTACTATTC 3' (SEQ ID NO. 43);                    -          5' GTATCGTTTCTTTCTTCATTCGC 3' (SEQ ID NO. 44);                     - (xxxvi)  5' GCTTGGGAGAATAATTTATCAG 3' (SEQ ID NO. 42);                      -          5' CCTCCTTCGTTAGTTGAATCCTC 3' (SEQ ID NO. 37);                     - (xxxvii) 5' GGACCAAGTTTACCATAATTC 3' (SEQ ID NO. 45);                       -          5' GTATCGTTTCTTTCTTCATTCGC 3' (SEQ ID NO. 44);                     - (xxxviii) 5' GGAGAATAATTTATCAGTTGGTC 3' (SEQ ID NO. 46);                    -           5' GTATCGTTTCTTTCTTCATTCGC 3' (SEQ ID NO. 44);                    - (xxxix)   5' CAAGGTATAAGCAGAAGAAAAC 3' (SEQ ID NO. 47);                     -           5' CGCACGCCCGGATTCTGTACTATC 3' (SEQ ID NO. 48);                   - (xl)      5' ATGTCTCAAATATCCTGGACTCAG 3' (SEQ ID NO. 49);                   -           5' CGCACGCCCGGATTCTGTACTATC 3' (SEQ ID NO. 48);                   - and                                                                         - (xli)     5' GTACTGGATAGATAGTGGAAGTC 3' (SEQ ID NO. 50);                    -           5' CACGGCCTTCTTCCTAACCCTCC 3' (SEQ ID NO. 51).             


10. A method according to claim 7 wherein the primers are selected fromthe group of primer pairs consisting of:

    (i)   5' GGTACTGGATAGATAGTGGA 3'                                                                        (SEQ ID NO. 2)                                         -        5' TCGCACGCCCGGATTCTGTA 3' (SEQ ID NO. 3);                           - (ii)  5' GAGATTCTGAAATTAATTGG 3' (SEQ ID NO. 27);                           -       5' CCTCCTTCGTTAGTTGAATCC 3' (SEQ ID NO. 59);                          - (iii)   5' GTTGGCTTATAGATTCTGAGC 3' (SEQ ID NO. 28);                        -        5' CAGTTCAAATTATTGTAGCC 3' (SEQ ID NO. 38);                          - (iv)  5' GAGATTCTGAAATTAATTGG 3' (SEQ ID NO. 27);                           -       5' CAGTTCAAATTATTGTAACC 3' (SEQ ID NO. 60);                           - (v)   5' GTTGGCTTATAGATTCTGAGC 3' (SEQ ID NO. 28);                          -        5' CCTCCTTCGTTAGTTGAATCC 3' (SEQ ID NO. 53);                         - (vi) 5' TAATTGGACATCAAGTTATAAAGC 3' (SEQ ID NO. 55);                        -      5' GGAAGATCCAAATAAGAATTATGG 3' (SEQ ID NO. 56);                        - (vii)   5' GGTTATTAAGATTCAAATTTCC 3' (SEQ ID NO. 29);                       -         5' CGAACTCTGATACGAGCTCCAAGC 3' (SEQ ID NO. 31);                     - (viii)    5' TCCCGTATCAGTTCGAGATTCTG 3' (SEQ ID NO. 30);                    -         5' CGAACTCTGATACGAGCTCCAAGC 3' (SEQ ID NO. 31);                     - (ix)   5' GTTCGAGATTCTGAAATTAATTGG 3' (SEQ ID NO. 39);                      -         5' CGAACTCTGATACGAGCTCCAAGC 3' (SEQ ID NO. 31);                     - (x)       5' TAATTGGACATCAAGTTATAAAGC 3' (SEQ ID NO. 53);                   -         5' CGAACTCTGATACGAGCTCCAAGC 3' (SEQ ID NO. 31);                     - (xi)   5' GGTTATTAAGATTCAAATTTCC 3' (SEQ ID NO. 29);                        -         5' CGAACTCTGATACGAGCTCCAAGC 3' (SEQ ID NO. 31);                     - (xii)    5' TCCCGTATCAGTTCGAGATTCTG 3' (SEQ ID NO. 30);                     -          5' GAATAGTACCTGGATCCAGATCCC 3' (SEQ ID NO. 33);                    - (xiii)       5' GGGATCAGGATCCAGGTACTATTC 3' (SEQ ID NO. 53);                                                   -          5' GAATAGTACCTGGATCCAGATC                                        CC 3' (SEQ ID NO. 33);                       - (xiv)   5' GGTTATTAAGATTCAAATTTCC 3' (SEQ ID NO. 29);                       -          5' GAATAGTACCTGGATCCAGATCCC 3' (SEQ ID NO. 33);                    - (xv)    5' TCCCGTATCAGTTCGAGATTCTG 3' (SEQ ID NO. 30);                      -      5' GGAAGATCCAAATAAGAATTATGG 3' (SEQ ID NO. 56);                        - (xvi)   5' GTTCGAGATTCTGAAATTAATTGG 3' (SEQ ID NO. 39);                     -      5' GGAAGATCCAAATAAGAATTATGG 3' (SEQ ID NO. 56);                        - (xvii)   5' GGTTATTAAGATTCAAATTTCC 3' (SEQ ID NO. 29);                      -      5' GGAAGATCCAAATAAGAATTATGG 3' (SEQ ID NO. 56);                        - (xviii)  5' ATTCGAGATTCTGAAATTAATTGG 3' (SEQ ID NO. 32);                    -          5' GAATAGTACCTGGATCCAGATCCC 3' (SEQ ID NO. 33);                    - (xix)    5' GATATTGGAGGGTTAGGAAGAAGG 3' (SEQ ID NO. 34);                    -          5' CTGTACAGTTCAAATTATTGTAACC 3' (SEQ ID NO. 35);                   - (xx) 5' GACTGATGAAATTATTAGTAAAGAGC 3' (SEQ ID NO. 36);                      -         5' CCTCCTTCGTTAGGTGAATCCTC 3' (SEQ ID NO. 37);                      - (xxi) 5' GGTACTGGATAGATAGTGGAAG 3' (SEQ ID NO. 57);                         -          5' CCAGAATCATAAGCTACTGTACC 3' (SEQ ID NO. 58);                     - (xxii)   5' GTCCCGTATCAGTTCGAGATTCTG 3'  (SEQ ID NO. 40);                   -          5' CCTCCTTCGTTAGTTGAATCCTC 3' (SEQ ID NO. 37);                     - (xxiii)   5' GGGATCAGGATCCAGGTACTATTC 3' (SEQ ID NO. 43);                   -          5' GTATCGTTTCTTTCTTCATTCGC 3' (SEQ ID NO. 44);                     - (xxiv)  5' GCTTGGGAGAATAATTTATCAG 3' (SEQ ID NO. 42);                       -          5' CCTCCTTCGTTAGTTGAATCCTC 3' (SEQ ID NO. 37);                     - (xxv) 5' GGACCAAGTTTACCATAATTC 3' (SEQ ID NO. 45);                          -          5' GTATCGTTTCTTTCTTCATTCGC 3' (SEQ ID NO. 44);                     - (xxvi) 5' GGAGAATAATTTATCAGTTGGTC 3' (SEQ ID NO. 46);                       -           5' GTATCGTTTCTTTCTTCATTCGC 3' (SEQ ID NO. 44);                    - (xxvii)   5' CAAGGTATAAGCAGAAGAAAAC 3' (SEQ ID NO. 47);                     -           5' CGCACGCCCGGATTCTGTACTATC 3' (SEQ ID NO. 48);                   - (xxviii)      5' ATGTCTCAAATATCCTGGACTCAG 3' (SEQ ID NO. 49);                                                  -           5' CGCACGCCCGGATTCTGTACT                                        ATC 3' (SEQ ID NO. 48);                      - and                                                                         - (xxix)     5' GTACTGGATAGATAGTGGAAGTC 3' (SEQ ID NO. 50);                   -           5' CACGGCCTTCTTCCTAACCCTCC 3' (SEQ ID NO. 51).             


11. A method according to claim 7 wherein the primer pair isGGTACTGGATAGATAGTGGA (Forward primer; SEQ ID NO. 2) andTCGCACGCCCGGATTCTGTA Reverse primer; SEQ ID NO. 3 ).
 12. A methodaccording to claim 7 wherein the primer pair is GAGATTCTGAAATTAATTGG(Forward primer; SEQ ID NO. 27) and CCTCCTTCGTTAGTTGAATCC (Reverseprimer; SEQ ID NO. 59).
 13. A method according to claims 2 or 8 whereinthe method includes a further step of testing for the viability and orthe infectivity of Cryptosporidium organisms in the sample.
 14. A kitfor the detection of Cryptosporidium isolates: the kit comprising atleast a probe or primer(s) selected from the nucleotide sequence definedin claim 1, which is capable of detecting Cryptosporidium isolates. 15.A kit according to claim 14 wherein the kit contains a primer pairselected from the primers defined in claims 9, 10, 11, or 12.